One of the current models for the coupling of the energy released from the mitochondrial electron transport chain to oxidative phosphory lation is an electrochemical gradient across, within, or at the surface-unstirred layer region of the inner membrane of the mitochondrion. The mechanism(s) of energy transduction is crucial in bioenergetics and has important clinical implications as well. Support is requested for the development of extrinsic, molecular, potential-sensitive probes in energy transducing membranes and for the testing of these probes in the cerebral cortex of the rat or gerbil. A series of polyene dyes and recently developed charge shift probes that appear to be electrochromic indicators of potential gradients will be the subject of the proposed work. Work will be carriedout using both intact mitochondria and submitochondrial particles. The long term goals of the work with mitochondrial membranes are: (1) the development of probes that are kinetically competent to follow the primary and secondary charge separation events that lead to electrochemical gradients and (2) a detailed understanding of the nature of the charge separation to which the probes are sensitive. Such knowledge will provide insight into the mechanism(s) responsible for energy coupling especially when the time course of the probe response is compared with that of electron transport components of energy coupling sites that may be involved in vectorial charge translocation. The kinetics work will be based on rapid mixing techniques in which dye-membrane suspensions are mixed with an appropriate substrate. The time course of the dye response will be followed by dual wavelength spectroscopy. A series of rapid mixing experiments are also designed to determine whether a probe is sensitive to transmembrane or to more localized potentials. NMR experiments are designed to provide insight into the location of the probes in the membrane; such location determines in part the nature of the charge separation to which the probes are sensitive. studies on the mechanisms by which probes respond to electrical gradients will be carried out in some cases. Selected probes will be tested for the ability to follow electrical activity changes in the cerebral cortex of the rat or gerbil during the normoxic to anoxic transition and to changes accompanying spreading depression initiated by topical KC1 application. The technicque of surface fluorimetry and reflectance will be used in this episode related work.